Chemical analysis of liquids, such as water, milk and biological fluids is often desirable or necessary for health maintenance and diagnostic treatment. Various compositions and elements to facilitate such analyses are known. Such materials generally comprise a reagent composition for determining the substance under analysis, identified herein as an "analyte". The analyte can be living cells, such as yeast cells, white blood cells or other living organisms, or a nonliving chemical substance. The reagent composition, upon interaction with the analyte, provides a detectable change (e.g. dye formation) which can be quantified in some manner.
The determination of specific hydrolytic enzymes in biological fluids can be useful for the diagnosis and treatment of certain diseases. It can also be useful for determining the presence of certain microorganisms because the metabolism of an organism is dependant upon the presence of a wide range of enzymes.
A number of analytical procedures have been developed whereby a substrate for an enzyme of interest is hydrolyzed to release a detectable moiety. These procedures use both colorimetric and fluorometric dyes. See, for example the assays described by Brown et al (J. Clin. Microbiology 21, p. 857, 1985) for pathogenic Nisseria spp., by Wasilauskas et al (J. Clin. Microbiology, 20, p. 1669, 1985) for Group A Streptococci, in the textbook edited by Norris (Methods of Microbiology, Vol. 9, Chapter 1, 1976) and by Kilian et al (Acta Path. Microbiol. Scand. B, 84, p. 245, 1976) for glycosidases.
Fluorometric assays are generally preferred because of generally greater sensitivity. However, known fluorometric assays are deficient in a number of ways. For example, E.P. application 122,148 (published Oct. 17, 1984) describes an assay for microorganisms using certain coumarin derivative as substrates which release dyes when the substrate is hydrolyzed. Although this assay is conducted at physiological pH for maximum biological activity, the pH is subsequently raised to 11 to obtain maximum fluorescence efficiency. Assays that require such a change in pH are not readily adaptable for highly automated analytical procedures.
U.S. Pat. No. 4,409,140 (issued Oct. 11, 1983 to Smith et al) describes an assay for proteolytic enzymes carried out at relatively lower pH, i.e. less than pH 8. The assay uses certain coumarin substrates which release a chromophore which fluoresces at 505 nm. At these relatively short wavelengths, however, spectral interferents (e.g. from hemoglobin and bilirubin) can be significant, severely limiting the sensitivity of the assay.
Other known assays require additional reagents, e.g. diazonium compounds, to give detectable moieties, as described in U.K. patent application No. 2,031,949 (published Apr. 30, 1980). Recently improved fluorescent unbelliferone derivatives have been described by Wolfbeis et al (Bull. Chem. Soc. Japan 58, p. 731, 1985) and used in an assay for acid phosphatase (Anal. Biochem. 143, p. 146, 1984). One of these dyes emits at 595 nm, but its absorption is at 505 nm where spectral interferents are a problem.
It is desirable to have fluorescent assays which are not subject to the problems associated with known assays, e.g. spectral interferents, and which can be readily used in automated analytical procedures.